1. Transgenic Corn as an Edible Vaccine
Joan Cunnick, Ph.D.
Dept. Animal Science
Iowa State University

2. Overview Vaccine history & types Transgenic plants
Edible vaccines at ISU
Future work

3. Small Pox 1250AD – a “childhood disease”
Edward Jenner 1796-observation pays off
Observation: Testing?
Jenner: Smallpox is Stemmed_____
"The first vaccination against smallpox was performed by Edward Jenner, English rural physician, in his apartment in the Chantry House, Berkeley Gloucestershire. Exudate from a cowpox pustule on the hand of a dairymaid, Sarah Nelmes, was inserted into scratches on the arm of eight-year-old James Phipps, May 14, The vaccination was effective, for two later attempts to induce infection with smallpox were unsuccessful. Proving and reproving his discovery, Jenner published his vaccination findings in Dispite opposition, vaccination became accepted practice during Jenner's lifetime." This painting by Robert A. Thom appeared in "Great Moments in Medicine" published by Parke Davis & Company, in The text quoted is from the same source. Both are reproduced with the permission of Parke Davis & Company.

4. One of the top 10 Medical Advances of 20th century
James Phipps - first person Edward Jenner vaccinated against smallpox
Ali Maow Maalin - last known case of naturally acquired smallpox (Somalia, 1977)
Luis Fermin - last person in the Western hemisphere to acquire paralytic poliomyelitis from wild poliovirus

5. The Golden Age of Microbiology
Louis Pasteur
“Did you ever observe to whom the accidents happen? Chance favors only the prepared mind.”
Attenuated vaccines

6. Important Milestones of 1900’s
“Its time we close the book on infectious disease”
-US Surgeon General, 1967
Small pox declared eradicated
1967
“Its time we close the book on infectious disease” US Surgeon General
Emerging Diseases

7. Some Current Vaccines Attenuated vaccines Killed/inactivated vaccines
oral polio (Sabin), MMR
Killed/inactivated vaccines
rabies, polio (Salk), Influenza
Subunit vaccines
HIB-Haemophilus influenzae type B
Hepatitis B

8. Some Current Vaccines Attenuated vaccines Killed/inactivated vaccines
oral polio (Sabin), MMR
Killed/inactivated vaccines
rabies, polio (Salk), Influenza
Subunit vaccines
HIB-Heamophilus influenzae type B
Hepatitis B

9. Injected Vaccines Induce Serum antibodies (Ab)
IgM
IgG
Protect from organisms in the blood and tissues

10. Why oral vaccine? Some organisms infect via mucosal tract
Gut pathogens
Respiratory pathogens
Ig A First line of defense-at mucosal surfaces
inhibit binding of bacteria
neutralize toxin

11. Why oral vaccine? Immunizing in mucosa favors IgA
ex: gut or intra-nasal
Cross protection of mucosal surfaces
Induces systemic immunity
Safer compared to injectable vaccines

12. Problems with oral vaccine
Ability to survive digestive enzymes
need for encapsulation
Large quantity of antigen needed or
Need for an appropriate adjuvant
1990’s - development of CT (Cholera toxin) & LT (E. coli heat labile toxin)

13. Biotech crops 1st generation: 2nd generation: 3rd generation:
Input-traits (agronomic benefits)
- Bt corn, RR soybean
2nd generation:
Output-traits (health and nutritional benefits)
- Vit A rice, Lycopene tomato
3rd generation:
New industry and pharmaceutical properties
- Vaccine corn, plastic or ethanol corn

14. Farm-aceuticals: why use plant as bioreactor ?
Safer
Plants carry no common mammalian pathogens
Animal viruses – Bovine viral diseases
Prions – Mad cow diseases
SV40 contamination in polio vaccine in 1960’s

15. Farm-aceuticals: why use plant as bioreactor ?
Safer
High-volume production
Seeds allow long-term protein storage and stability
11 million children each year die from
Pneumonia, Diarrhea, Measles, Malaria, Malnutrition
Source: WHO Fact Sheet No 178, 1998

16. Rationale for using corn
Major staple food and feed worldwide
Most tolerated plant for both humans and animals
Can be fed raw
Yield
Seeds allow long-term protein storage and stability
Established infrastructure for production and protein extraction
Possible low-cost production and administration of proteins
Genetic transformation is routine in ISU

17. Targeting of protein expression
Gene construction
Promoter determines site of protein expression
Constitutive
Callus/Leaves
Seed specific
Germ
Starch storage
Natural encapsulation?
Ubiquitin promoter
27 kD  -zein promoter

18. E. coli heat labile toxin (LT): A-B Toxin
B=binding
A= active toxin

19. E. coli heat labile toxin (LT): A-B Toxin
B=binding
Remove A subunit
LT-B 82% homologous
with CT-B

20. Rationale for using LT-B
Ideal model antigen
Strong immunogen
Induces strong mucosal and serum responses when orally administered.
Potent adjuvant

21. Immunogenicity of corn expressed LT-B
BALB/c mice fed with ….
….corn meal pellets expressing μg LTB/g corn at days 0, 3, 7, 21.

22. ELISA to measure Ab Enzyme linked immunosorbent assay
LT-B protein
from T. plants
Ganglioside GM1

23. ELISA to measure Ab Enzyme linked immunosorbent assay
Source of antibodies:
serum
mucosal (fecal pellets)
Mouse Antibodies
LT-B protein
from T. plants
Ganglioside GM1

24. ELISA to measure Ab Enzyme linked immunosorbent assay
Anti-mouse IgG-HRP
ENZYME
Mouse Antibodies
LT-B protein
from T. plants
Ganglioside GM1

25. ELISA to measure Ab Enzyme linked immunosorbent assay
Anti-mouse IgG-HRP
Substrate
ENZYME
Mouse Antibodies
Product
LT-B protein
from T. plants
Ganglioside GM1

26. ELISA to measure Ab more Ab = more enzyme = more color
Anti-mouse IgG-HRP
Substrate
ENZYME
Mouse Antibodies
ENZYME
Product
ENZYME
Product
LT-B protein
from T. plants
ENZYME
Product
Product
Ganglioside GM1

27. Does Corn LT-B induce a serum & mucosal Ab response
Sample collection
Feed
4 mice per group
Transgenic groups
20 µg LT-B
2 µg LT-B
0.2 µg LT-B
0.02 µg LT-B
Non-transgenic groups
Non-transgenic (nt)
Collected serum and fecal samples weekly

30. Effect of CT and LT on gut:carcass ratio of mice
Patent mouse assay:
Effect of CT and LT on gut:carcass ratio of mice

31. Antibodies protect from whole toxin
Mice challenged with 25 μg LT or CT

32. Anti-LT-B IgA is produced at distant sites: Lung

33. Future Promise of LT-B vaccine
B=binding
Replace A subunit with protein of interest
Influenza?
SARS?
PRRS? ?

34. Future Promise of LT-B vaccine
B=binding
Replace A subunit with protein of interest
Influenza?
SARS?
PRRS?
GFP

35. If biotech crops contaminate corn used for food…
Will the vaccine still be effective?

36. Daily vs. intermittent LT-B feeding Affect of exposure on vaccine response
Treatments:
Intermittent Daily
20 µg LT-B 20 µg LT-B
2 µg LT-B 2 µg LT-B
0.2 µg LT-B 0.2 µg LT-B
non-transgenic (nt) non-transgenic (nt)
4 mice were included in each group for a total of 32 mice.

37. Serum IgG Daily vs. intermittent LT-B feeding

38. Serum IgG Affect of exposure on vaccine response

39. Summary Vaccines are important public health initiatives
Plant derived vaccines can utilize the best of subunit vaccines, but are safer
Transgenic corn LT-B is a potent immunogen
Safety considerations need to be examined as vaccine is being developed

40. Colleagues Kan Wang (ISU, Agronomy) Rachel Chikwamba Sule Karaman
April Beyer
Sarah Rahn

41. Opportunities for High School & Middle School Students
Microbiology High School Workshop
Nov 14, :45AM - 3:10PM
Joan Cunnick
Science in Iowa (Middle School; April date) &
Visiting Scientist Program (All Grades-throughout year)
College of Agriculture
Topics change each year
Mickie Franks